Enantioselective immunoaffinity extraction for simultaneous determination of optically active bufuralol and its metabolites in human plasma by HPLC

Abstract

A combined method of immunoaffinity extraction with high-performance liquid chromatography has been developed for the enantioselective determination of bufuralol and its metabolites in human plasma. The antibodies having high affinity toward the asymmetric center at the C-1 position of bufuralol and its 1′-oxidized metabolites and low affinity to their antipodes were elicited by immunization of rabbits with immunogens, (1 R)- and (1 S)-1′-oxobufuralol O-carboxymethyl oxime-bovine serum albumin conjugates, respectively. 0.5 ml Of the immunoaffinity adsorbent (7.6 mg·ml −1 for anti-(1 S)-antibody and 28.8 mg·ml −1 for anti-(1 R)-antibody) prepared by immobilization of an antibody was capable of retaining up to 1 μg of ( R)- and ( S)-bufuralol and up to 500 ng of other metabolites. The adsorbates were recovered quantitatively by elution with methanol–10 mM ammonium acetate buffer (pH 5) (95:5, v/v) without any interfering peaks on the high-performance liquid chromatogram. The proposed method was evaluated to be useful for the simultaneous determination of optically active bufuralol and its metabolite in plasma with acceptable recovery and precision.